The glycoprotein autotaxin (ATX) is a member of the ecto-nucleotide pyrophosphatase and phosphodiesterase (eNPP) family of proteins, which was initially cloned and purified in this laboratory as a tumor motility-stimulating factor. Recently, ATX was found to be identical to serum lysophospholipase D (LPLD). In addition, in experimental tumor models, ATX has been shown to enhance tumorigenesis, invasiveness, metastasis, and angiogenesis. We are studying the mechanisms by which ATX stimulates invasion, metastasis, and angiogenesis and how these biological functions relate to its enzymatic activity. Utilizing a series of point mutations within the LPLD active site, we found that motility stimulation requires intact enzyme activity. In fact, ATX motility stimulation appears to depend upon the production of LPA or S1P, depending upon which EDG receptors are present on the cell surface. We are continuing to study structure-function relationships within the ATX molecule. We have a number of new point mutations, including several within the enzyme active site and 6 that are part of a collaborative effort with Dr. Suk Woo Nam at the Catholic University of Korea who is looking at ATX mutations occurring in breast cancer patients. Among the active site point mutations that we have constructed are mutations that completely lack enzyme or motility activity and a mutation that retains both activities at an intermediate level. The mutations found in patients are now all in plasmids, and we are currently characterizing these mutant ATX's. A second major focus for our group has been to study how ATX regulates cell behavior and how it interacts with other essential cytokines and growth factors. Utilizing morpholino-oligonucleotides (ATX-specific vs mismatch controls), which reduce translation rather than transcription, we have decreased ATX secretion in two carcinoma cell lines to levels that are undetectable by immunoblot. One very important effect of the ATX knockdown in the human ovarian carcinoma cell line SKOV3 is significantly reduced motility response to multiple chemoattractants. We plan to determine if ATX knockdowns have a similar inhibitory effect with in vitro invasion assays.